Distinct blood transcriptomic signature of treatment in latent tuberculosis infected individuals at risk of developing active disease.

Although only a small fraction will ever develop the active form of tuberculosis (ATB) disease, chemoprophylaxis treatment in latent TB infected (LTBI) individuals is an effective strategy to control pathogen transmission. Characterizing immune responses in LTBI upon chemoprophylactic treatment is important to facilitate treatment monitoring, and thus improve TB control strategies. Here, we studied changes in the blood transcriptome in a cohort of 42 LTBI and 8 ATB participants who received anti-TB therapy. Based on the expression of previously published gene signatures of progression to ATB, we stratified the LTBI cohort in two groups and examined if individuals deemed to be at elevated risk of developing ATB before treatment (LTBI-Risk) differed from others (LTBI-Other). We found that LTBI-Risk and LTBI-Other groups were associated with two distinct transcriptomic treatment signatures, with the LTBI-Risk signature resembling that of treated ATB patients. Notably, overlapping genes between LTBI-Risk and ATB treatment signatures were associated with risk of progression to ATB and interferon (IFN) signaling, and were selectively downregulated upon treatment in the LTBI-Risk but not the LTBI-Other group. Our results suggest that transcriptomic reprogramming following treatment of LTBI is heterogeneous and can be used to distinguish LTBI-Risk individuals from the LTBI cohort at large.

Prenatal chromosome microarray: 'The UK experience'. A survey of reporting practices in UK genetic services (2012-2019).

BACKGROUND: The value of chromosome microarray (CMA) in the prenatal detection of significant chromosome anomalies is well-established. To guide the introduction of this technique in routine clinical practice, the Joint Committee on Genomics in Medicine developed national UK guidelines for reporting prenatal CMA in 2015. OBJECTIVE: To evaluate the UK experience of utilising prenatal CMA. METHOD: A 36-item survey was distributed to all UK clinical genetics services (n = 23) in March 2019 requesting information pertaining to experience since diagnostic testing commenced and current practice (March 2018 to March 2019). RESULTS: Eighteen UK genetics services currently offer prenatal CMA. A total of 14,554 tests had been performed. A pathogenic copy number variant was identified in 7.8% of tests overall, though the diagnostic rate increased to 8.4% in the final year of the survey. Variants of uncertain significance (VUS) were reported in 0.7% of tests, and 'actionable' incidental findings in 0.12%. CONCLUSION: Diagnostic rate has improved over time, while reporting of VUS has decreased. Reviewing survey responses at a national level highlights variation in testing experience and practice, raising considerations both for future guideline development and implementation of other novel techniques including prenatal whole exome sequencing.

Abnormal nuchal translucency followed by normal microarray analysis is associated with placental pathology-related complications.

OBJECTIVE: Identify placental pathology-related complications, labor and neonatal outcomes in pregnancies complicated by pathological nuchal translucency (NT) with normal microarray analysis. METHODS: A retrospective study in which all women with singleton pregnancy who demonstrated NT above 3 mm and a normal microarray analysis were matched to women with normal NT and a normal microarray analysis (2013-2019) in a single tertiary academic center. The following placental pathology-related parameters were measured: preeclampsia, oligohydramnios, suspected intrauterine growth restriction, abnormal Doppler studies or small for gestational age (SGA) neonates. The primary outcome was defined as a composite of complications related to placental pathology including preeclampsia and SGA neonate. Secondary outcomes were labor complications and neonatal morbidity. RESULTS: A total of 185 women were included in the study: of them, 47 presented an abnormal NT (study group) and 138 presented normal NT (controls). Groups did not significantly differ in baseline characteristics. Regarding primary outcome, all placental-related complications frequencies were higher in the study group, with a composite rate of 17.02% versus 6.52% in controls (p = 0.042%). Secondary outcomes did not differ between groups. CONCLUSIONS: Abnormal NT measurement presented in pregnancies with normal fetal microarray analysis is associated with higher rates of placental-related complications.

Personalized prenatal genomic testing: Couples' experience with choice regarding uncertain and adult-onset findings from chromosomal-microarray-analysis.

BACKGROUND: Chromosomal-microarray-analysis (CMA) can identify variants of uncertain clinical significance, susceptibility-loci for neurodevelopmental conditions, and risk for adult-onset conditions. We explored choices made by couples undergoing prenatal CMA, their understanding of these findings, reasons for and against receiving them, and whether they believe parents or professionals should decide which are disclosed. METHODS: Semi-structured interviews were conducted with women (n = 27) or their partners (n = 15) during the week following prenatal CMA testing and analyzed using grounded theory. RESULTS: Over half the interviewees (55%) recalled at least two of the three types of CMA results they chose whether to receive. Sixty-four percent found the choice simple, whereas 36% found it difficult. All participants could clearly explain their choices, which were based on the perceived actionability and psychological impact of the information. Sixty percent viewed their choice favorably, whereas ~21% would have preferred clinicians to decide for them. More women than men, and more decisive than indecisive participants supported parental choice. CONCLUSION: Overall, expectant parents can make informed choices about which uncertain findings about their fetuses they wish to receive, and value the opportunity to tailor results to their values and wishes. Arguments presented provide the basis for a decision-aid tool for expecting parents.

Digital quantitative assessment of PD-L1 using digital spatial profiling.

The assessment of programmed death 1 ligand 1 (PD-L1) expression by Immunohistochemistry (IHC) is the US Food and Drug Administration (FDA)-approved predictive marker to select responders to checkpoint blockade anti-PD-1/PD-L1 axis immunotherapies. Different PD-L1 immunohistochemistry (IHC) assays use different antibodies and different scoring methods in tumor cells and immune cells. Multiple studies have compared the performance of these assays with variable results. Here, we investigate an alternative method for assessment of PD-L1 using a new technology known as digital spatial profiling. We use a previously described standardization tissue microarray (TMA) to assess the accuracy of the method and compare digital spatial profiler (DSP) to each FDA-approved PD-L1 assays, one LDT assay and three quantitative fluorescence assays. The standardized cell line Index tissue microarray contains 10 isogenic cells lines in triplicates expressing various ranges of PD-L1. The dynamic range of PD-L1 digital counts was measured in the ten cell lines on the Index TMA using the GeoMx DSP assay and read on the nCounter platform. The digital method shows very high correlation with immunohistochemistry scored with quantitative software and with quantitative fluorescence. High correlation of PD-L1 digital DSP counts were seen between rows on the same Index TMA. Finally, experiments from two Index TMAs showed reproducibility of DSP counts were independent of variable slide storage time over a three-week period after antibody labeling but before collection of cleaved tags. In summary, DSP appears to have quantitative potential comparable to quantitative immunohistochemistry. It is possible that this technology could be used as a PD-L1 protein measurement system for companion diagnostic testing for immune therapy.

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR.

CDH17 Is a More Sensitive Marker for Gastric Adenocarcinoma Than CK20 and CDX2.

CONTEXT: -CDH17, which is expressed in the intestinal epithelium, is a novel oncogene involved in tumor invasion and metastasis. A panel consisting of cytokeratin (CK) 7, CD20, and CDX2 antibodies is typically used to diagnose gastrointestinal adenocarcinomas. However, studies have shown that CDH17 is a highly specific marker for gastrointestinal adenocarcinoma and may be important in clinical diagnosis. OBJECTIVE: -To evaluate the sensitivity and specificity of CDH17, CK20, and CDX2 antibodies in neoplastic tissues, with emphasis on colon, stomach, and esophageal gastrointestinal lineage. DESIGN: -Immunohistochemistry was performed with CDH17, CK20, and CDX2 antibodies on formalin-fixed, paraffin-embedded tissue microarrays from normal (n = 26) and neoplastic (n = 884) tissues. RESULTS: -CDH17 immunostaining was positive in 97.3% (145 of 149) of colon adenocarcinomas, whereas CK20 and CDX2 stained positively in 88.6% (132 of 149) and 93.3% (139 of 149), respectively. In metastatic colon cancers, CDH17, CK20, and CDX2 positive staining was observed in 90.6% (29 of 32), 59.4% (19 of 32), and 81.3% (26 of 32) of cases, respectively. In stomach adenocarcinomas, CDH17 positively stained 64.0% (112 of 175) of tissues, compared to CK20 and CDX2, where staining was observed in only 24.6% (43 of 175) and 46.9% (82 of 175), respectively. In esophageal adenocarcinomas, CDH17, CK20, and CDX2 stained 38.7% (12 of 31), 25.8% (8 of 31), and 29% (9 of 31) of specimens, respectively. Low or no expression was observed in other neoplastic tissues, except pancreatic cancers, where CDH17 displayed higher expression than CK20 and CDX2. CONCLUSIONS: -CDH17 is a specific and more sensitive marker in the gastrointestinal tract than CK20 and CDX2. CDH17 may be especially valuable when gastrointestinal tumors are suspected in cancers of unknown primary.

High scavenger receptor class B type I expression is related to tumor aggressiveness and poor prognosis in breast cancer.

Scavenger receptor class B type I (SR-BI) has been linked to the development and progression of breast cancer. However, its clinical significance in breast cancer remains unclear. Here, we evaluated SR-BI expression in a well-characterized breast cancer tissue microarray by immunohistochemistry. High SR-BI expression was observed in 54 % of all breast cancer cases and was significantly associated with advanced pTNM stage (P = 0.002), larger tumor size (P = 0.023), lymph node metastasis (P = 0.012), and the absence of ER (P = 0.014). The Kaplan-Meier survival analysis revealed that patients with high SR-BI expression had significantly shorter overall survival (OS) (P = 0.004). Moreover, multivariate analysis with adjustment for other prognostic factors confirmed that SR-BI was an independent prognostic factor for patient outcome (P = 0.017). Overall, our study demonstrated that high SR-BI expression was related to conventional parameters indicative of more aggressive tumor type and may serve as a new prognostic marker for poor clinical outcome in human breast cancer.

Protease Activated Receptors 1 and 2 Correlate Differently with Breast Cancer Aggressiveness Depending on Tumor ER Status.

Experimental models implicate protease activated receptors (PARs) as important sensors of the proteolytic tumor microenvironment during breast cancer development. However, the role of the major PARs, PAR-1 and PAR-2, in human breast tumors remains to be elucidated. Here, we have investigated how PAR-1 and PAR-2 protein expression correlate with established clinicopathological variables and patient outcome in a well-characterized cohort of 221 breast cancer patients. Univariable and multivariable hazard ratios (HR) were estimated by the Cox proportional hazards model, distant disease-free survival (DDFS) and overall survival by the Kaplan-Meier method, and survival in different strata was determined by the log-rank test. Associations between PARs and clinicopathological variables were analyzed using Pearson's χ2-test. We find that PAR-2 associates with DDFS (HR = 3.1, P = 0.003), whereas no such association was found with PAR-1 (HR = 1.2, P = 0.6). Interestingly, the effect of PAR-2 was confined to the ER-positive sub-group (HR = 5.5, P = 0.003 vs. HR = 1.2 in ER-negative; P = 0.045 for differential effect), and PAR-2 was an independent prognostic factor specifically in ER-positive tumors (HR = 3.9, P = 0.045). On the contrary, PAR-1 correlated with worse prognosis specifically in the ER-negative group (HR = 2.6, P = 0.069 vs. HR = 0.5, P = 0.19 in ER-positive; P = 0.026 for differential effect). This study provides novel insight into the respective roles of PAR-1 and PAR-2 in human breast cancer and suggests a hitherto unknown association between PARs and ER signaling that warrants further investigation.

HER2/neu testing in gastric cancer by immunohistochemistry: assessment of interlaboratory variation.

CONTEXT: Immunohistochemical (IHC) testing for HER2/neu is becoming the standard of care for guiding adjuvant treatment of gastric carcinoma with trastuzumab. OBJECTIVE: To assess interlaboratory variation in IHC staining and interpretation across multiple laboratories. DESIGN: A tissue microarray consisting of 45 cores from 28 gastric cancers was distributed to 37 laboratories for HER2/neu assessment. The IHC results were compared against expert scores at an academic institution and correlated with in situ hybridization results from the originating specimen. Interlaboratory agreement was calculated using Cohen κ statistic. RESULTS: The survey demonstrated several variations in IHC methods, including the primary antibodies in use. There was excellent agreement among laboratories in HER2/neu(+) (IHC 3(+)) cases (κ = 0.80 ± 0.01) and very good agreement among laboratories in HER2/neu(-) (IHC 0 or 1(+)) cases (κ = 0.58 ± 0.01). Less agreement was observed among laboratories when scoring equivocal (IHC 2(+)) cases (κ = 0.22 ± 0.01). Sensitivity and specificity of HER2/neu IHC were 99% and 100%, respectively, when measured against expert review and consensus score as a reference standard. CONCLUSIONS: There is substantial interlaboratory agreement in the interpretation of HER2/neu IHC despite variability in protocols. Although HER2/neu IHC is a highly sensitive and specific test, primary antibody selection may significantly affect IHC results. Furthermore, gastric tumors require a unique scoring system and expertise in interpretation. Intratumoral heterogeneity has a significant effect on HER2/neu scoring by IHC. Ongoing quality assurance exercises among laboratories will help ensure optimized HER2/neu testing.

Accurate data processing improves the reliability of Affymetrix gene expression profiles from FFPE samples.

Formalin fixed paraffin-embedded (FFPE) tumor specimens are the conventionally archived material in clinical practice, representing an invaluable tissue source for biomarkers development, validation and routine implementation. For many prospective clinical trials, this material has been collected allowing for a prospective-retrospective study design which represents a successful strategy to define clinical utility for candidate markers. Gene expression data can be obtained even from FFPE specimens with the broadly used Affymetrix HG-U133 Plus 2.0 microarray platform. Nevertheless, important major discrepancies remain in expression data obtained from FFPE compared to fresh-frozen samples, prompting the need for appropriate data processing which could help to obtain more consistent results in downstream analyses. In a publicly available dataset of matched frozen and FFPE expression data, the performances of different normalization methods and specifically designed Chip Description Files (CDFs) were compared. The use of an alternative CDFs together with fRMA normalization significantly improved frozen-FFPE sample correlations, frozen-FFPE probeset correlations and agreement of differential analysis between different tumor subtypes. The relevance of our optimized data processing was assessed and validated using two independent datasets. In this study we demonstrated that an appropriate data processing can significantly improve the reliability of gene expression data derived from FFPE tissues using the standard Affymetrix platform. Tools for the implementation of our data processing algorithm are made publicly available at http://www.biocut.unito.it/cdf-ffpe/.

High chondroitin sulfate proteoglycan 4 expression correlates with poor outcome in patients with breast cancer.

Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified in melanoma cells, has been reported to be expressed in breast cancer cells. This study was performed to examine the expression and significance of CSPG4 in a cohort of breast cancer patients. Immunohistochemical analysis of CSPG4 was performed on tissue microarrays constructed from tissue specimens from 240 breast cancer patients. CSPG4 staining was correlated with clinical and pathological characteristics, overall survival (OS), and disease recurrence. Contradicting to a previous report, our results showed that high CSPG4 expression was not related to triple-negative status of breast cancer patients. The Kaplan-Meier method showed that high CSPG4 expression was significantly associated with shorter time to recurrence (TTR). Patients with high CSPG4 expression had poorer OS and shorter TTR in a multivariate survival analysis after adjustment for stage, tumor grade, expression of estrogen receptor and progesterone receptor, and HER2 overexpression. This study showed that high CSPG4 expression correlates with disease recurrence and OS in breast cancers.

Expression of pim-1 in tumors, tumor stroma and tumor-adjacent mucosa co-determines the prognosis of colon cancer patients.

Provirus integration site for Moloney murine leukemia virus (pim-1) is a proto-oncogene that is linked to the development and progression of several cancers. In this study, we evaluated pim-1 expression in tumors, tumor stroma and tumor-adjacent mucosa together as an independent prognostic factor for colon cancer patients. The study included 343 colon cancer patients. Immunohistochemical staining was used to detect pim-1. Multivariate cox regression for disease-free survival (DFS) were used to identify independent prognostic factors. Analytic hierarchy process (AHP) was used to calculate the weight of pim-1 in tumors, tumor stroma and tumor-adjacent mucosa in order to obtain a Pim-1 total score (PTS) for recurrence and survival. Kaplan-Meier DFS curves and OS curves for patients with different pim-1 expression levels were compared using the log-rank test. In this study, four independent prognostic factors were identified for colon cancer patients: pim-1 expression in tumors, tumor stroma, tumor-adjacent mucosa, as well as tumor stage. It has been established that clinical stage is an important prognostic factor for colon cancer patients. However, PTS can identify the patients who are likely to recur not only in the whole radical excision group but also within each stage of this group. Based on the results of this study we can conclude that the PTS combined with clinical staging system may be a better predictor of colon cancer patients' prognosis than using the clinical stage system alone. ClinicalTrials.gov Number: ChiCTR-PRCH-12002842.

Low PIP4K2B expression in human breast tumors correlates with reduced patient survival: A role for PIP4K2B in the regulation of E-cadherin expression.

Phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase ß (PIP4K2B) directly regulates the levels of two important phosphoinositide second messengers, PtdIns5P and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2]. PIP4K2B has been linked to the regulation of gene transcription, to TP53 and AKT activation, and to the regulation of cellular reactive oxygen accumulation. However, its role in human tumor development and on patient survival is not known. Here, we have interrogated the expression of PIP4K2B in a cohort (489) of patients with breast tumor using immunohistochemical staining and by a meta-analysis of gene expression profiles from 2,999 breast tumors, both with associated clinical outcome data. Low PIP4K2B expression was associated with increased tumor size, high Nottingham histological grade, Ki67 expression, and distant metastasis, whereas high PIP4K2B expression strongly associated with ERBB2 expression. Kaplan-Meier curves showed that both high and low PIP4K2B expression correlated with poorer patient survival compared with intermediate expression. In normal (MCF10A) and tumor (MCF7) breast epithelial cell lines, mimicking low PIP4K2B expression, using short hairpin RNA interference-mediated knockdown, led to a decrease in the transcription and expression of the tumor suppressor protein E-cadherin (CDH1). In MCF10A cells, knockdown of PIP4K2B enhanced TGF-ß-induced epithelial to mesenchymal transition (EMT), a process required during the development of metastasis. Analysis of gene expression datasets confirmed the association between low PIP4K2B and low CDH1expression. Decreased CDH1 expression and enhancement of TGF-ß-induced EMT by reduced PIP4K2B expression might, in part, explain the association between low PIP4K2B expression and poor patient survival.

Guidelines and considerations for conducting experiments using tissue microarrays.

Tissue microarrays (TMAs) represent a powerful method for undertaking large-scale tissue-based biomarker studies. While TMAs offer several advantages, there are a number of issues specific to their use which need to be considered when employing this method. Given the investment in TMA-based research, guidance on design and execution of experiments will be of benefit and should help researchers new to TMA-based studies to avoid known pitfalls. Furthermore, a consensus on quality standards for TMA-based experiments should improve the robustness and reproducibility of studies, thereby increasing the likelihood of identifying clinically useful biomarkers. In order to address these issues, the National Cancer Research Institute Biomarker and Imaging Clinical Studies Group organized a 1-day TMA workshop held in Nottingham in May 2012. The document herein summarizes the conclusions from the workshop. It includes guidance and considerations on all aspects of TMA-based research, including the pre-analytical stages of experimental design, the analytical stages of data acquisition, and the postanalytical stages of data analysis. A checklist is presented which can be used both for planning a TMA experiment and interpreting the results of such an experiment. For studies of cancer biomarkers, this checklist could be used as a supplement to the REMARK guidelines.

Large-scale independent validation of the nuclear factor-kappa B p65 prognostic biomarker in prostate cancer.

PURPOSE: Over the last decade, we and others have uncovered a robust association between the nuclear localisation of nuclear factor-kappa B (NF-κB) p65, prostate cancer (PCa) aggressiveness and biochemical recurrence (BCR). Our goal was to validate these results in a large independent cohort of PCa patients who underwent radical prostatectomy. EXPERIMENTAL DESIGN: A set of 1826 fully annotated prostate cancers treated by radical prostatectomy were analysed in a tissue microarray (TMA) format for NF-κB p65 immunohistochemistry-based protein expression. We performed standard Cox proportional hazard regression models for follow-up data, bootstrap procedure for model internal validation, Harrell's concordance index for model discrimination and graphical assessment of predicted versus actual outcomes for model calibration. RESULTS: We observed a significant association between an increase in the nuclear frequency of NF-κB p65 and Gleason score (P<0.001), overall BCR (P<0.001) and development of metastases (P=0.001). NF-κB was found to be an independent predictor of BCR (P<0.001, Cox regression). However its contribution to the predictive accuracy of a multivariate model, which included preoperative PSA, Gleason score, extraprostatic extension, lymph node invasion, seminal vesicle involvement and surgical margin status, was modest. CONCLUSIONS: Our study offers validating results linking NF-κB p65 with disease progression using a large cohort of European men. However, the contribution of NF-κB to a post-surgical predictive model appears modest. Further validating work should focus on evaluating the contribution of NF-κB p65 in pre-treatment models.

Loss of SPINK1 expression is associated with unfavorable outcomes in urothelial carcinoma of the bladder after radical cystectomy.

BACKGROUND: We assessed the association of serine protease inhibitor Kazal type I (SPINK1) expression with clinicopathologic outcomes in urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC). MATERIALS AND METHODS: Tissue microarrays comprising 438 consecutive UCB patients treated with RC between 1988 and 2003 and 62 cases of normal urothelium controls were evaluated for SPINK1 protein expression by immunohistochemistry (IHC). Semiquantitative evaluation was performed by 2 pathologists blinded to clinical outcomes (loss of expression: <50% cells or intensity 0-2). RESULTS: In normal urothelium, SPINK1 expression was noted in umbrella cells of 32 of 62 controls (52%); 254 RC patients (57.9%) exhibited loss of SPINK1 expression. Loss of SPINK1 expression was significantly associated with higher pathologic stages (P = 0.002) and presence of lymph node metastasis (P = 0.04). At a median follow-up of 130 months (IQR: 98.4), loss of SPINK1 expression was associated with an increased risk of disease recurrence (P = 0.02) and cancer-specific mortality (P = 0.03). On multivariable analysis that adjusted for the effects of standard clinicopathologic parameters, SPINK1 was not an independent predictor of disease recurrence (P = 0.09) or cancer-specific mortality (P = 0.12). CONCLUSIONS: Over half of UCB patients treated with RC exhibit loss of SPINK1 expression. Loss of SPINK1 correlates with features of biologically aggressive UCB. Although SPINK1 expression did not have independent prognostic value in RC patients, it may serve as a biomarker for tumor staging and may be useful as an adjunct in clinical decision-making.

Tissue-based Alzheimer gene expression markers-comparison of multiple machine learning approaches and investigation of redundancy in small biomarker sets.

BACKGROUND: Alzheimer's disease has been known for more than 100 years and the underlying molecular mechanisms are not yet completely understood. The identification of genes involved in the processes in Alzheimer affected brain is an important step towards such an understanding. Genes differentially expressed in diseased and healthy brains are promising candidates. RESULTS: Based on microarray data we identify potential biomarkers as well as biomarker combinations using three feature selection methods: information gain, mean decrease accuracy of random forest and a wrapper of genetic algorithm and support vector machine (GA/SVM). Information gain and random forest are two commonly used methods. We compare their output to the results obtained from GA/SVM. GA/SVM is rarely used for the analysis of microarray data, but it is able to identify genes capable of classifying tissues into different classes at least as well as the two reference methods. CONCLUSION: Compared to the other methods, GA/SVM has the advantage of finding small, less redundant sets of genes that, in combination, show superior classification characteristics. The biological significance of the genes and gene pairs is discussed.

Neutrophil gelatinase-associated lipocalin (NGAL) predicts response to neoadjuvant chemotherapy and clinical outcome in primary human breast cancer.

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.

BTN3A2 expression in epithelial ovarian cancer is associated with higher tumor infiltrating T cells and a better prognosis.

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR=0.651, p=0.006 and HR=0.642, p=0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR=1.355 p=0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.